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RNA Biology
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Probing the closed-loop model of mRNA translation in
living cells
a
b
c
d
Stuart K Archer , Nikolay E Shirokikh , Claus V Hallwirth , Traude H Beilharz & Thomas
Preiss
ae
a
Genome Biology Department; The John Curtin School of Medical Research (JCSMR); The
Australian National University; Acton (Canberra), Australian Capital Territory, Australia
b
Moscow Regional State Institute of Humanities and Social Studies; Ministry of Education of
Moscow Region; Kolomna, Moscow Region, Russia
c
The Gene Therapy Research Unit; Children's Medical Research Institute; Westmead
(Sydney), New South Wales, Australia
Click for updates
d
Department of Biochemistry & Molecular Biology; Monash University; Clayton (Melbourne),
Victoria, Australia
e
Victor Chang Cardiac Research Institute; Darlinghurst (Sydney), New South Wales, Australia
Published online: 31 Mar 2015.
To cite this article: Stuart K Archer, Nikolay E Shirokikh, Claus V Hallwirth, Traude H Beilharz & Thomas Preiss (2015) Probing
the closed-loop model of mRNA translation in living cells, RNA Biology, 12:3, 248-254, DOI: 10.1080/15476286.2015.1017242
To link to this article: http://dx.doi.org/10.1080/15476286.2015.1017242
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BRIEF COMMUNICATION
RNA Biology 12:3, 248--254; March 2015; Published with license by Taylor & Francis
Probing the closed-loop model of mRNA
translation in living cells
Stuart K Archer1, Nikolay E Shirokikh2, Claus V Hallwirth3, Traude H Beilharz4, and Thomas Preiss1,5,*
1
Genome Biology Department; The John Curtin School of Medical Research (JCSMR); The Australian National University; Acton (Canberra), Australian Capital Territory, Australia;
Moscow Regional State Institute of Humanities and Social Studies; Ministry of Education of Moscow Region; Kolomna, Moscow Region, Russia; 3The Gene Therapy Research Unit;
Children’s Medical Research Institute; Westmead (Sydney), New South Wales, Australia; 4Department of Biochemistry & Molecular Biology; Monash University;
Clayton (Melbourne), Victoria, Australia; 5Victor Chang Cardiac Research Institute; Darlinghurst (Sydney), New South Wales, Australia
2
Downloaded by [Australian National University] at 19:28 06 September 2015
Keywords: cap-poly(A) synergy, eukaryotic translation, mRNA closed-loop, polysomes, ribosomal recycling
The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB,
features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not
well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked
cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a
proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for
PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNAspecific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of
gene expression.
Introduction
The first vizualization of ribosomes by electron microscopy (EM)
almost 60 y ago immediately sparked an interest in the topology of
their arrangement into polysomes.1 A string of EM studies focusing
on eukaryotic endoplasmic reticulum-bound ribosomes then
revealed that membrane-attached polysomes commonly form circular or hairpin-like arrangements.2 Membrane surface effects and a
tendency of mRNA to curve through the ribosome were thought to
favor these phenomena, however, the potential for ribosome
‘recycling’ within looped polysomes was also noted and experimentally substantiated.3,4 At the time, no specific mechanism for such
ribosome transfer in cis could be discerned.
The demonstration of functional synergy between the mRNA
50 cap and 30 poly(A) tail for translation in the early nineties then
sparked renewed interest in mRNA looping.5 Intense research,
largely using in vitro translation (IVT) systems,6-9 led to the
establishment of the widely accepted ‘closed-loop’ model of
translation initiation, which posits that mutual interactions of
the cap-binding eukaryotic initiation factor eIF4E, the adaptor
protein eIF4G (together forming eIF4F in yeast), and the poly
(A)-binding protein (PAB) hold the 50 and 30 ends of mRNA in
close proximity and promote recruitment of the small ribosomal
subunit to the mRNA 50 end.10-12 Atomic force microscopy
allowed observation of closed-loop structures after mixing eIF4E,
eIF4G, PAB and a model mRNA in vitro.13 While short-lived
IVT reactions typically only initiate a few rounds of translation,
longer-lived continuous exchange IVT systems can assemble
steady-state polysomes, the latter again providing evidence for
formation of hairpin-like assemblies and ribosome recycling.14
However, this did not require the presence of either a cap or
poly(A) tail on the mRNA, suggesting that additional 50 to 30 or
ribosome stacking interactions may suffice for mRNA looping
and ribosome recycling.15 Nevertheless, strong support for the
cap-to-tail closed-loop is drawn from the fact that its constituent
interactions serve as a major convergence point for multiple
mechanisms of translational control.11,16
Given these divergent observations, we set out to directly test the
prevalence of the cap-to-tail closed-loop, by affinity-isolation of
eIF4F-PAB1 complexes after formaldehyde cross-linking of live
cells. We found for multiple mRNAs under steady-state conditions
that the closed-loop was prevalent for eIF4F-bound molecules, while
it was only a minor configuration for PAB1-bound transcripts. We
further observed mRNA-specific differences in the extent of closedloop formation that persisted after a reset of cellular translation.
Results and Discussion
We used yeast lines carrying combinations of tagged versions
of translation initiation factors eIF4E, eIF4G and PAB1
© Stuart K Archer, Nikolay E Shirokikh, Claus V Hallwirth, Traude H Beilharz, and Thomas Preiss
*Correspondence to: Thomas Preiss; Email: [email protected]
Submitted: 01/15/2015; Accepted: 01/15/2015
http://dx.doi.org/10.1080/15476286.2015.1017242
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s)
have been asserted.
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(Supplementary Fig. 1). These were grown to exponential
growth phase in liquid culture, snap-cooled and crosslinked with
3% formaldehyde to create in vivo ‘snapshots’ of polysomes at
steady-state. Specifically, formaldehyde treatment has been
shown to rapidly fortify translation initiation intermediates such
that they can be co-purified with polysomes.17,18 Cross-linked
cells were lysed by a bead-beating method into non-denaturing
buffer and complexes assembled around a given initiation factor
carrying a C-terminal Protein A (ProA) tag were purified on IgG
beads. Recovery of proteins and mRNAs was monitored by western blotting and RT-qPCR, respectively. Controlled RNase I
digestion was used to allow for selective co-purification of only
factor-associated portions of mRNAs (Fig. 1A). Complete, nonfractionated lysate was processed in parallel and used as a reference for the RT-qPCR experiments. As expected, eIF4E, eIF4G
and PAB1 all co-purified with each other, which was only marginally affected by RNase treatment (Supplementary Fig. 2).
The rationale of our experimental approach was to use the
extent of the interaction between these factors and the opposing
ends of mRNAs as a proxy for closed-loop formation. Several
types of complex assemblies could formally explain a co-purification of both mRNA ends in such complexes. However, given the
scarcity of eIF4G19 and the well-characterized affinities of eIF4E,
eIF4G and PAB for each other (the latter requiring RNA binding
to interact with eIF4G20), the cap-to-tail closed-loop is the most
parsimonious explanation of such an observation. We first
focused on SSC1 mRNA, an abundant transcript with precisely
mapped 30 and 50 extremities.21 Five qPCR primer pairs (2 close
to the ends and 3 internal) were designed, each spanning no
more than 75 bp (Fig. 1B; see Materials and Methods for selection criteria). eIF4E, eIF4G and PAB1 were quantitatively isolated from RNase-treated cell lysates on IgG beads, and
fragments of the SSC1 mRNA were analyzed for co-enrichment
relative to RNase-digested input controls by RT-qPCR. eIF4E
and eIF4G cooperatively bind stably to capped mRNAs22 and
thus affinity purification of either eIF4F constituent resulted in
similar co-enrichment of both ends of the SSC1 mRNA relative
to the central regions (Fig. 1B; eIF4E: brown line; eIF4G: green
line). The degree of 30 end enrichment in eIF4E IP (4E:30 ) relative to the 50 end (4E:50 ) gave a lower bound on the prevalence
of the closed-loop conformation in eIF4F-bound SSC1 mRNA
of »35%. The C-terminal tag on the eIF4E fusion protein also
contained a Calmodulin Binding Protein (CBP) moiety (Supplementary Fig. 1), allowing it to be successfully purified using a
completely different solid phase and affinity group (Calmodulinagarose). This again showed both 50 and 30 end enrichment of
SSC1 mRNA (Supplementary Fig. 3A), demonstrating independent replication. By contrast, affinity purification of PAB1
resulted in strong enrichment of the 30 end relative to the central
regions, with a selective but much weaker enrichment of the 50
end (»6% relative to that of the 30 end). This was much less
than the corresponding enrichment of the 30 end seen when
immunoprecipitating eIF4E/4G (Fig. 1B; purple line). These
results were reiterated using N-terminally tagged PAB1 to
exclude the possibility that the C-terminal tag location interferes
with the closed-loop conformation (Supplementary Fig. 3B).
Collectively, these results indicate that the closed-loop configuration is detectable for both, eIF4F-bound and PAB1-bound SSC1
mRNA in vivo. Surprisingly, however, the closed-loop proportion was much higher for eIF4F-bound than for PAB1-bound
SSC1 mRNA.
Next, we performed 3 independent replicates of tagged eIF4E
and PAB1 purifications (Fig. 2A) and analyzed a further 16
mRNAs, obtaining results generally equivalent to those with
SSC1 (Fig. 2B). (In the
case of the PAB1 mRNA,
the high 50 -UTR association with PAB1 likely
indicates that it is subject
to autoregulation in yeast,
via a poly(A) tract in its 50
UTR, as has been demonstrated in other eukaryotes.23) The enrichment
of 30 sequences co-purifying with eIF4E (4E:30 )
varied between different
mRNAs much more than
that of 50 sequences
(4E:50 ). This was not due
to differences in the distances of qPCR primers
from the poly(A) tail,
Figure 1. (A) Overall experimental strategy. Each yeast line used encoded a recombinant version of either PAB1, eIF4G
because this would have
or eIF4E bearing an affinity tag with a Protein A moiety. The corresponding proteins were affinity-purified in the presequally affected 30 enrichence of ribonuclease. (B) Top: qPCR primers were designed against various regions of a target transcript to test for copurification. Bottom: Enrichment of SSC1 transcript regions with eIF4E, eIF4G and PAB1, by RT-qPCR. IP / Input was norment from PAB1 purificamalized to the average across all 5 qPCR assays in SSC1. Error bars represent 95% confidence intervals from 3 qPCR
tions (PAB:30 ); on the
replicates.
contrary we observed that
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Figure 2. For figure legend, see page 251.
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PAB:30 variability could not explain the majority of the variance
of 4E:30 (Supplementary Fig. 4). Further, the variance of 4E:30
between different mRNAs was large, with the highest (RSP20)
being approximately 8-fold higher that the lowest (GUS1), while
30 qPCR target-to-poly(A) tail distances were all similar (Supplementary Fig. 5). We could not detect any strong correlation
between closed-loop prevalence and global datasets on translational efficiency,24 ORF length, poly(A) tail length (Harrison, P.
et al., unpublished results), or mRNA half-life25 (although a
weak inverse relationship was detected with the latter (Supplementary Fig. 6)).
When cells were glucose-starved for 10 minutes followed by
re-feeding, to collapse and reassemble polysomes (Fig. 2C), there
was only a marginal increase in mean 4E:30 (pD0.045, one-tailed
t-test), and mRNAs generally retained the relative differences in
their levels of 4E:30 seen before the disruption (Fig. 2D). Thus,
these differences are not a function of differences in the relative
time spent in translation of these mRNAs, since they remain in
place after a global translational reset. Only the TDH1 mRNA
showed a significant increase over this trend; this might be linked
to its drastic transcriptional upregulation by starvation26
(Fig. 2D). During the actual glucose starvation, the extremities
of most mRNAs co-IP with less specificity relative to the internal
mRNA regions (Supplementary Fig. 7). This is likely due to the
sequestration of mRNA, eIF4E, eIF4G and PAB into EGP (4E/
4G/PAB) bodies, which are protein-dense RNA-containing granules that form by 10 minutes into glucose starvation to sequester
and safeguard mRNAs.27 Formaldehyde would heavily crosslink
mRNA with protein in these bodies, likely hindering efficient
RNase digestion of mRNA bodies. (Only 2 mRNAs, ACO2 and
RCT3, exhibited normal eIF4E co-IP profiles during glucose starvation; possibly linked to the fact that expression is sensitive to
glucose availability in both cases.39,40 Overall, these findings
indicate that the extent of closed-loop formation is governed by
mRNA-intrinsic determinants rather than by nutritional status
or translation state.
We have shown here, using a representative set of mRNAs,
that cap-to-tail closed-loop interactions are formed in living yeast
cells, thus underpinning a central tenet of the contemporary
model of eukaryotic translation initiation with direct experimental evidence. The extent of observed closed-loop formation was
shown to differ between mRNAs, and the differences were maintained after complete interruption and restart of bulk protein
synthesis. Transcriptome-wide surveys of the closed-loop would
be warranted to more fully explore whether it correlates with, or
could perhaps serve as a surrogate measurement of, other parameters of mRNA function.
An unexpected result was that the extent of observable cap-totail closed-loop differed markedly between complexes purified
via tags on eIF4E/4G versus PAB1. PAB1 is an abundant protein
while eIF4G is relatively scarce in yeast cells.19,28 Further, PAB1
more readily co-purifies with polysomes than do eIF4F subunits.29 Thus it is reasonable to view PAB1-bound mRNAs as representative of the whole population, while eIF4F-bound
molecules form a specific subset active in closed-loop assembly.
As the average polysome occupancy of mRNA molecules is more
than 70% in exponentially growing yeast cells30 this suggests
that, at least once established, polysomes can be maintained without a permanent cap-to-tail closed-loop. Thus cap-to-tail interactions might only prevail during a transient phase of mRNA
activation and/or transcript-specific regulation events that serve
to establish full polysome association. Consistent with this concept it was shown that mRNA decay, a process requiring access
to the ends of mRNA, is already initiated on actively translating
polysomes,31,32 and also that ongoing translation initiation in
established polysomes is resistant to loss of eIF4G.15,33 Future
work should employ time-resolved analyses to investigate how
mRNAs can alternate between different factor-bound vs.
unbound, and closed vs. open states.
What is the potential for looped polysomes in the absence of
permanent stable cap-to-tail interactions? PAB is known to maintain bridging interactions to translation termination at the stop
codon as well as 60S subunit joining at the start codon,11,30,31,34
and other less well understood interactions may facilitate ribosome recycling in cis.14,15,35 Nonetheless, we observed that the
cap-to-tail closed-loop occurs in significant amounts in stably
eIF4F-bound mRNAs. The findings presented here inform and
modify our views on how polysome topology and post-transcriptional control mechanisms acting on the cap-to-tail closed-loop
can converge in the regulation of gene expression.
Materials and Methods
Yeast lines
The eIF4G1:TEV-ProA / eIF4G2-line was generated in the
w303a background (leu2-3,112; trp1-1; can1-100; ura3-1; ade21; his3-11,15) by in situ tagging eIF4G1 at the C-terminus using
a Protein A cassette with the KanMX6 (G418R) selectable marker
in w303a, and mating this with w303a cells in which eIF4G2
Figure 2 (See previous page). (A) Quantitative isolation of protein A-tagged eIF4E (top) or PAB1 (bottom) by IP using IgG affinity chromatography, and
detection of co-purifying eIF4G. Input lanes: 1£, 0.5£, 0.25£, 0.125£ of lysate equivalents. FT: Flow-through, 1£ equivalent. IP: Immunoprecipitated fraction, 1£ equivalent. (B) Co-enrichment of mRNA extremities with eIF4E and PAB1 (average of 3 biological replicates, error bars correspond to standard
error of the mean). Values were normalized to the average 3’ enrichment (for PAB1) or 5’ enrichment (for eIF4E) across all transcripts. Blue dashed lines:
transcript extremities. (C) Collapse and restoration of polysomes after glucose starvation (10 minutes) and re-feeding (5 minutes) of eIF4E::ProA yeast
cultures. Representative UV-absorbance traces (sedimentograms) of crosslinked cytoplasmic extracts fractionated through sucrose gradients. Signal to
the right of the 80S monosome peak (indicated) corresponds to translated (polysomal) mRNAs. (D) Correlation between the closed-loop status (4E:3’) of
transcripts with or without starvation / re-feeding. Error bars denote standard error of the mean (3 biological replicates). Only TDH1 showed significant
change (corrected p D 0.006).
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had been knocked out using a KanMX6 replacement cassette.
Diploids were sporulated and haploid clones screened by PCR and
protein gel blotting. The eIF4E::CBP::ProA line was generated by
amplifying a TAP tag cassette flanked by eIF4E 30 sequences19 and
transforming into an isogenic (w303a) line using selection on
histidine dropout media. The PAB1::ProA line was generated by
transforming the pYM8 construct19,36 with flanking PAB1 30
sequences into the w303a line and selecting on G418 media.
To generate a C-terminally c-myc tagged PAB protein in the
eIF4E::TAP line, a construct was made using the pYM4 tagging
construct36 containing the kanMX6 selectable marker as a template, and amplifying using primers containing flanking sequences derived from the 30 end of the PAB1 ORF and 30 UTR. The
resulting vector was transformed into the eIF4E::TAP line
(described above) and selected on growth medium containing
G418. Colonies were screened by western blotting for c-myctagged PAB1, and the region sequenced to check for non-silent
mutations. To generate a C-terminally GFP-tagged eIF4E protein in the PAB::ProA and eIF4G::ProA lines (described above),
this line was transformed with a eIF4E::GFP (HIS+) in situ tagging cassette, which had been amplified from an existing eIF4E::
GFP yeast line37 by PCR. Colonies were screened by protein gel
blotting and PCR and sequenced to check for non-silent mutations. To generate a line in which PAB1 was N-terminally tagged
with ProA, the pYM-N5 construct38 was amplified by PCR using
primers containing flanking PAB1 sequences and transformed
into a eIF4E::GFP line. Colonies were selected on nourseothricin
(Werner BioAgents) and checked for proper construct integration
by western blotting and sequencing. The construct confers a copper-activated promoter to PAB1 as well as the N-terminal ProA
moiety and thus was grown in the presence of 100 mM CuSO4
at all times (although titration experiments with CuSO4 did not
indicate that growth rates fell below that of the wild-type at most
other CuSO4 concentrations). All lines are summarized in Supplementary Fig. 1.
Yeast culture, in vivo crosslinking and lysis
Yeast were grown to mid-log phase (to an optical density at
600 nm of 0.7–0.8) under standard growth conditions (30 C
with shaking). Liquid cultures were flash-cooled by addition of
25% (w/v) of crushed ice, briefly stirring, and addition of 10%
(v/v) cold 30% (w/v) PFA within 20–30 seconds of the ice. The
culture was transferred to polypropylene centrifuge tubes and
allowed to crosslink for 8 minutes at 4 C before transferring to a
pre-cooled rotor and spinning at 6,000 £g for 5 minutes. The
supernatant was poured off, and 18–19 minutes after addition of
PFA, cells were resuspended in cold HBB buffer (20 mM
HEPES pH7.4, 100 mM KCl, 2 mM MgCl2) with 0.25 M glycine to quench PFA and transferred to pre-weighed 50 ml conical
tubes. Cells were pelleted by cold centrifugation, resuspended in
HBB and pelleted again. The cell pellet was aspirated and
weighed, and resuspended in 1 ml HBB-DRP (HBB supplemented with 0.5 mM DTT, 1£ RNase OUT (Life Technologies) and 1£ Complete EDTA-free mini protease inhibitor
cocktail from Roche) per gram of wet cell weight. This cell suspension was snap frozen by dripping into liquid nitrogen, and
252
the frozen material homogenized by 2 rounds of 1 minute of
shaking at 27 Hz in a canister with steel ball-bearings. The canister was kept cold by intermittently submerging in liquid nitrogen. The resultant powder was stored frozen until needed for
downstream steps.
Immunoprecipitation of ProA-tagged proteins
ProA-tagged proteins (or eIF4E tagged with the TAP tag)
were immunoprecipitated using magnetic beads (epoxy-activated
Dyna beads, Life Technologies) conjugated with human IgG
(Sigma) according to the manufacturer’s instructions. The
amount of beads required to deplete >85% of the TAP-tagged
PAB from 50 mg of grindate was empirically determined for
each batch of beads (typically the equivalent of »7.5 mg dry
beads) and that amount was used for precipitation of all proteins.
About 100 mg of grindate was weighed into a 1.5 ml tube over
liquid nitrogen and placed on ice »1 minute. Cold buffer A
(25 mM HEPES pH7.4, 140 mM KCl, 1.8 mM MgCl2, 0.1%
IPEGAL CA-630) supplemented with 0.5 mM DTT, 35 mM
NaCl, 100 U/ml RNase OUT and 1£ Complete EDTA-free
mini protease inhibitor cocktail was added (4£ v/w) and vortexed at high speed until the grindate was thawed and resuspended, then for a further 5 seconds and placed on ice. Cell
debris was pelleted (5 minutes at 12,000£ g) and the supernatant
clarified in a second round of centrifugation (10 minutes at
12,000£ g). The absorbance at 260 nm was measured on a
Nanodrop spectrophotometer (Thermo Scientific). 3U of RNase
I (Ambion) was added per A260 unit (Absorbance at 260 nm £
volume in ml) of lysate. An aliquot of the digestion reaction was
retained as the input sample. The volume of the remainder was
measured, and then it was added to the predetermined amount
of aspirated IgG beads. The bead-lysate mixture was rotated at
ambient temperature (22 C) for 30 minutes, and the input sample was placed alongside it. Both were then placed on ice, and the
beads were collected on a magnetic rack at 4 C. Beads were
rinsed briefly in 1 ml ice-cold wash buffer, then collected and
washed twice more for 5 minutes each with rotating. Meanwhile,
the bead supernatant and the input sample were aliquoted into 2
samples each (for RNA and protein isolation) and frozen. After
the second wash, beads were resuspended in the original volume
(measured after removal of the input sample) and aliquots of the
slurry removed for protein analysis, while the remainder (the
major portion of beads) was kept for RNA isolation. All bead
slurry aliquots were collected on a magnet and aspirated before
freezing for later use.
For a typical batch of TAP-calmodulin pulldown, 50 ml of 4%
calmodulin-agarose slurry (Sigma-Aldrich) were used per 100 mg of
yeast grindate prepared as described above. Upon melting of the
grindate, 4 volumes (w/v) of cold buffer CB (25 mM HEPESKOH pH 7.6, 50 mM KCl, 3 mM MgCl2, 3.5 mM CaCl2,
4 mM DTT, 0.5 mM EDTA, 0.5% glycerol, 0.05% Triton-X100)
were added and the solution was immediately supplemented with
1 U/ml RNase inhibitor (RiboLock, Thermo Scientific). The resultant mixture was then processed as it is described for the IgG immunoprecipitation protocol above, until the resin binding step. The
clarified, RNase I-supplemented lysate was incubated at room
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temperature for 25 minutes to allow RNA digestion and then added
to the calmodulin-agarose beads pre-equilibrated with CB buffer in
a 1.5 ml microcentrifuge tube. Binding was performed for 30
minutes at 4 C with slow rotation. Beads were pelleted by centrifugation at 3000£ g for 3 minutes, aspirated and washed 3 times with
1 ml of CW buffer (CB buffer with 100 mM KCl) at 4 C for 5
minutes with slow rotation. To release the calmodulin-bound material, 200 ml of CE buffer (25 mM HEPES-KOH pH 7.6, 100 mM
KCl, 3 mM MgCl2, 4 mM DTT, 1 mM EDTA, 15 mM EGTA,
1% glycerol, 0.1 U/ml RNaseOUT RNase inhibitor, 0.05% Triton-X100) were added to the washed and aspirated beads and the
mixture was incubated at 4 C for 30 minutes with slow rotation.
Crosslink reversal and protein and RNA isolation
RNA was isolated from beads and lysate using a hot acid phenol
method, which also reverses formaldehyde-mediated crosslinks,
modified to reduce SDS precipitation and phenol carryover. To frozen, aspirated beads or lysate (<50 ml) 400 ml of Buffer SETG (1%
SDS, 10 mM EDTA, 10 mM Tris from 1M stock of pH 7.4,
10 mM Glycine from 1M stock of pH 2.5) was added and mixed.
400 ml of 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (pH »4.5,
Sigma Aldrich) were added, the lids securely closed and the mixture
shaken at 65 C, 1300 rpm on a thermomixer for 45 minutes. The
mixture was then centrifuged at 15,000£ g at room temperature for
5 minutes, and the aqueous phase removed and precipitated by adding 20 mg of glycogen, 0.1 volume of 3M sodium acetate and 2.5
volumes of ethanol. After precipitation at ¡20 C for at least
3 hours, RNA was pelleted at 16,000£ g 25 minutes, the RNA pellet washed twice in 70% ethanol and dried at 37 C for at least 30
minutes in a sterile hood to evaporate residual phenol. Dried RNA
was thoroughly resuspended by repeated pipetting and vortexing in
1 mM sodium citrate, pH 6.0, and assessed by measuring the absorbance spectrum on a Nanodrop spectrophotometer.
cDNA synthesis and qPCR
RNA for cDNA synthesis was treated with Turbo DNase (Life
Technologies) in the presence of RNase Inhibitors (RNase In,
Life Technologies) and the DNase inactivated using DNase inactivation resin according to the manufacturer’s instructions.
cDNA was synthesized from RNA using a cocktail of gene-specific primers (all reverse primers for the qPCR reactions, Supplementary Table 1).
High throughput qPCR
High throughput qPCR was performed in a Fluidigm 9216-well
reaction chip (Fluidigm PN BMK-M-96.96) according to the manufacturer’s instructions for qPCR using EvaGreen dsDNA detection
dye (Protocol PN 100-1208-A2, Fluidigm). Briefly, cDNA samples
including IP samples and serial dilutions were pre-amplified in a 96well plate using all primers in all assays by limited PCR, followed by
an Exonuclease I digestion to degrade the preamplification primer
mixture. The chip was loaded with primer pairs and samples to
include at least 3 technical replicates for each assay / sample combination, as well as a dilution curve for the calculation of qPCR primer
pair efficiency for each assay. Primers used in the qPCR are as listed
in Supplementary Table 1. The chip was loaded into a Fluidigm
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Biomark HD system and subjected to thermocycling. Captured
fluorescence signals were then filtered for reaction quality and used
to calculate the threshold-cycle (Ct) value using the Fluidigm BioMark software. These were exported into R to adjust for qPCR reaction efficiency, statistical analysis and production of plots.
Western blotting
Protein samples were heated in 1£ Laemmli buffer and
loaded onto denaturing polyacrylamide gels (NuPAGE bis-tris
protein gels, Novex), electrophoresed and electroblotted onto
Protran PVDF membranes (Amersham). Bulk protein loading
was visualized on the membrane using Red Alert Western stain
(Millipore) following the manufacturer’s instructions, and photographed on a Gel-doc system. The stain was subsequently washed
out using transfer buffer containing 20% methanol. For immunoblotting, the membranes were immersed in PBST (PBS with
0.1% Tween-20) for washing steps (3 £ 5 minutes at room temperature), while for blocking and antibody incubations (1 hour
each, at 4 C), 5% non-fat milk in PBST was used. eIF4G1 was
detected using a rat primary antibody raised against amino acids
542-823 of eIF4G1. Protein-A tagged eIF4E, eIF4G and PAB1
were detected using HRP-conjugated rabbit IgG (Abcam, cat#
ab106045). Myc-tagged and GFP-tagged proteins were detected
using mouse monoclonal antibodies (Roche products
11667149001 and 11814460001 respectively). Actin was
detected using Horseradish Peroxidase (HRP)-conjugated rabbit
polyclonal antibody (Abcam, ab106045). Mouse and rat primary
antibodies were detected using Santa Cruz HRP-conjugated antibodies (sc-2005 and sc-2006, respectively). Detection was performed by mixing equal volumes of 0.02% H2O2 with 2.4 mM
luminol / 0.4 mM coumaric acid (all in 100 mM Tris-HCl pH
8.5) and adding the mixture to the membrane. HRP-driven
chemiluminescence signal was visualized and digitized on an
ImageQuant LAS 4000 system (GE Healthcare).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgements
High-throughput qPCR was performed with the assistance of
Stephanie Palmer and the Australian Cancer Research Foundation Biomolecular Resource Facility at the Australian National
University.
Funding
Funding was provided by the Australian Research Council
under grant number DP1300101928. TP was supported by an
NHMRC Senior Research Fellowship. NES was supported by a
Go8 European Fellowship.
Supplemental Material
Supplemental data for this article can be accessed on the
publisher’s website.
RNA Biology
253
Downloaded by [Australian National University] at 19:28 06 September 2015
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